Mitogen-activated protein kinases (MARK) are major effector proteins, involved in cell proliferation, differentiation and apoptosis. Alterations in MARK signaling lead to development of various patho-physiological conditions. Signaling through MAPKs is predominantly controlled by growth factors, in particular, EGF. EGF receptor (EGFR) activation triggers a network of signal transduction events along with accelerated endocytosis of EGF-receptor complexes. It is proposed that endocytic trafficking regulates the intensity and duration of MARK signaling, but the mechanisms of this regulation are not understood. Moreover, both characteristics (signaling duration and intensity) have been shown to depend on the proper function of MARK scaffold proteins, yet their role in signal propagation has not been fully elucidated. The main goal of this project is to identify the role of endocytosis in activation of the MARK cascade and to define the regulatory mechanisms of endocytosis and signaling that are critical for cancer development and progression. We will analyze how EGFR activation and endocytic trafficking provides spatial and temporal control of MARK pathway signaling. We will use a quantitative method of high-resolution microscopy, fluorescence resonance energy transfer (FRET), that has been developed in our lab and has been previously used to demonstrate the presence of EGFR signaling complexes in endosomes of living cells. Here we propose a novel siRNA Knock Down And Rescue (KDAR) strategy to identify involvement of endocytic machinery in MARK activation. A number of reagents and new methodologies developed in our lab will allow us to characterize the localization of several components of MAPK1/2 module in time in living cells under conditions of physiological expression levels. The research proposed here will provide new insight into the specific mechanisms of regulation of the signal-transduction events by endocytic trafficking. The purpose of this research is to understand the regulatory mechanisms of the molecular cascade that affect tumor progression. Results from this study will define requirements of this intracellular signaling pathway for normal cell function, which will lead to development of new diagnostic markers work described in the Research Proposal will be conducted at the University of Colorado Health Sciences Center, Aurora Colorado